GLUTATHIONE TRANSPORT IN IMMORTALIZED HLE CELLS AND EXPRESSION OF TRANSPORT IN HLE CELL POLY(A)-INJECTED XENOPUS-LAEVIS OOCYTES( RNA)

PURPOSE. To determine reduced glutathione (GSH) transport in cultured human lens epithelial cells (HLE-B3) and plasma membrane vesicles and to study the expression of GSH transport in Xenopus laevis oocytes inj ected with poly(A)(+) RNA from HLE-B3 cells. METHODS. ConfIuent HLE-B3 cells pretreated with 10 mM DL-buthionine sulfoximine and 0.5 mM aciv icin were used in GSH uptake studies. The uptake of S-35-GSH was perfo rmed for 30 minutes in either NaCl medium (Na+-containing) or choline chloride medium (N+-free) at 37 degrees C and 4 degrees C. The molecul ar form of S-35 uptake was determined by high-performance liquid chrom atography. GSH uptake kinetics were studied in acivicin and buthionine sulfoximine-treated HLE-B3 cells in NaCl medium in the concentration range 0.01 mu M to 50 mM. The transport of GSH and the effect of Na+ o n uptake also were determined in mixed plasma membrane vesicles from H LE-B3 cells. In oocyte expression studies, HLE-B3 poly(A)(+) RNA was i njected into X. laevis oocytes and GSH uptake experiments were perform ed 3 days after injection. The uptake of S-35-GSH and GSH efflux rates ; were determined in HLE-B3 poly(A)(+) RNA-injected oocytes. RESULTS. No significant difference was found in the uptake of 1 mM GSH +/- aciv icin (17.7 +/- 4.3 versus 15.7 +/- 1.4 picomoles/min(-1) per 10(6) cel ls). However, GSH uptake was significantly lower in Na+-free medium co mpared with Na+-containing medium (10.3 +/- 0.7 versus 16.8 +/- 0.9 pi comoles/min(-1) per 10(6) cells; P < 0.01). GSH uptake in NaCl medium was carrier mediated. GSH uptake showed partial sodium dependency from 5 mu M to 5 mM GSH in mixed plasma membrane vesicles from HLE-B3 cell s. Oocytes injected with HLE-B3 poly(A)(+) RNA expressed uptake and ef fIux of GSH. Uptake showed partial Na+ dependency at various GSH conce ntrations. The efflux rates were approximately 30-fold higher than tho se in water-injected oocytes (0.48 +/- 0.03 versus 0.016 +/- 0.005 (na nomoles per hour(-1) per oocyte, respectively). The molecular form of uptake in cultured cells and in oocyte studies was predominantly as in tact GSH. CONCLUSIONS. HLE-B3 cells and plasma membrane vesicles trans ported GSH by a carrier-mediated process. HLE-B3 poly(A)(+) RNA inject ed X laevis oocytes expressed GSH transport. GSH uptake was partially Na+ dependent in all systems. HLE-B3 cells offer a useful model for ch aracterizing GSH transport and for studying its regulatory role in the etiology of cataracts.